Differential ordering of the protein backbone and side chains during protein folding revealed by site-specific recombinant infrared probes

The time scale for ordering of the polypeptide backbone relative to the side chains is a critical issue in protein folding. The interplay between ordering of the backbone and ordering of the side chains is particularly important for the formation of β-sheet structures, as the polypeptide chain searches for the native stabilizing cross-strand interactions. We have studied these issues in the N-terminal domain of protein L9 (NTL9), a model protein with mixed α/β structure. We have developed a general approach for introducing site-specific IR probes for the side chains (azide) and backbone ((13)C═(18)O) using recombinant protein expression. Temperature-jump time-resolved IR spectroscopy combined with site-specific labeling enables independent measurement of the respective backbone and side-chain dynamics with single residue resolution. We have found that side-chain ordering in a key region of the β-sheet structure occurs on a slower time scale than ordering of the backbone during the folding of NTL9, likely as a result of the transient formation of non-native side-chain interactions.     

Side-chain and backbone ordering in a polypeptide

We report results from multicanonical simulations of polyglutamic acid chains of length of ten residues. For this simple polypeptide we observe a decoupling of backbone and side-chain ordering in the folding process. While the details of the two transitions vary between the peptide in gas phase and in an implicit solvent, our results indicate that, independent of the specific surroundings, upon continuously lowering the temperature side-chain ordering occurs only after the backbone topology is completely formed.     

Side-chain and backbone ordering in homopolymers

In order to study the relation between backbone and side-chain ordering in proteins, we have performed multicanonical simulations of deka-peptide chains with various side groups. Glu(10), Gln(10), Asp(10), Asn(10), and Lys(10) were selected to cover a wide variety of possible interactions between the side chains of the monomers. All homopolymers undergo helix-coil transitions. We found that peptides with long side chains that are capable of hydrogen bonding, i.e., Glu(10), and Gln(10), exhibit a second transition at lower temperatures connected with side-chain ordering. This occurs in the gas phase as well as in solvent, although the character of the side-chain structure is different in each case. However, in polymers with short side chains capable of hydrogen bonding, i.e., Asp(10) and Asn(10), side-chain ordering takes place over a wide temperature range and exhibits no phase transition-like character. Moreover, non-backbone hydrogen bonds show enhanced formation and fluctuations already at the helix-coil transition temperature, indicating competition between side-chain and backbone hydrogen bond formation. Again, these results are qualitatively independent of the environment. Side-chain ordering in Lys(10), whose side groups are long and polar, also takes place over a wide temperature range and exhibits no phase transition-like character in both environments. Reasons for the observed chain length threshold and consequences from these results for protein folding are discussed.     

Statistical mechanics of protein allostery: roles of backbone and side-chain structural fluctuations

A statistical mechanical model of allosteric transition of proteins is developed by extending the structure-based model of protein folding to cases that a protein has two different native conformations. Partition function is calculated exactly within the model and free-energy surfaces associated with allostery are derived. In this paper, the model of allosteric transition proposed in a previous paper [Proc. Natl. Acad. Sci. U.S.A 134, 7775 (2010)] is reformulated to describe both fluctuation in side-chain configurations and that in backbone structures in a balanced way. The model is applied to example proteins, Ras, calmodulin, and CheY: Ras undergoes the allosteric transition between guanosine diphosphate (GDP)-bound and guanosine triphosphate (GTP)-bound forms, and the model results show that the GDP-bound form is stabilized enough to prevent unnecessary signal transmission, but the conformation in the GTP-bound state bears large fluctuation in side-chain configurations, which may help to bind multiple target proteins for multiple pathways of signaling. The calculated results of calmodulin show the scenario of sequential ordering in Ca(2+) binding and the associated allosteric conformational change, which are realized though the sequential appearing of pre-existing structural fluctuations, i.e., fluctuations to show structures suitable to bind Ca(2+) before its binding. Here, the pre-existing fluctuations to accept the second and third Ca(2+) ions are dominated by the side-chain fluctuation. In CheY, the calculated side-chain fluctuation of Tyr106 is coordinated with the backbone structural change in the β4-α4 loop, which explains the pre-existing Y-T coupling process in this protein. Ability of the model to explain allosteric transitions of example proteins supports the view that the large entropic effects lower the free-energy barrier of allosteric transition.     

Fine grained sampling of residue characteristics using molecular dynamics simulation

In a fine-grained computational analysis of protein structure, we investigated the relationships between a residue's backbone conformations and its side-chain packing as well as conformations. To produce continuous distributions in high resolution, we ran molecular dynamics simulations over a set of protein folds (dynameome). In effect, the dynameome dataset samples not only the states well represented in the PDB but also the known states that are not well represented in the structural database. In our analysis, we characterized the mutual influence among the backbone ?,ψ angles with the first side-chain torsion angles (χ₁) and the volumes occupied by the side-chains. The dependencies of these relationships on side-chain environment and amino acids are further explored. We found that residue volumes exhibit dependency on backbone 2° structure conformation: side-chains pack more densely in extended β-sheet than in α-helical structures. As expected, residue volumes on the protein surface were larger than those in the interior. The first side-chain torsion angles are found to be dependent on the backbone conformations in agreement with previous studies, but the dynameome dataset provides higher resolution of rotamer preferences based on the backbone conformation. All three gauche^?, gauche⁺, and trans rotamers show different patterns of ?,ψ dependency, and variations in χ₁ value are skewed from their canonical values to relieve the steric strains. By demonstrating the utility of dynameomic modeling on the native state ensemble, this study reveals details of the interplay among backbone conformations, residue volumes and side-chain conformations.     

Theory of volume phase transitions of side-chain liquid crystalline gels

We present a mean field theory to describe volume phase transitions of side-chain liquid crystalline gels. Three different uniaxial nematic phases (N(1), N(2), and N(3)) are defined by using orientational order parameter S(m) of side-chain liquid crystals (mesogens) and S(b) of backbone chains. We derive the free energy for the three nematic phases of side-chain liquid crystalline gels dissolved in isotropic solvents and calculate the swelling curve of the gel, the order parameters of a backbone chain and of side-chain liquid crystals, and the deformation of the gel as a function of temperature and an electric field. We find isotropic-nematic (N(1), N(2), and N(3)) and N(1)N(2) phase transitions of the gels, depending on the interaction between a backbone chain and a side-chain liquid crystal.     

Protein folding using fragment assembly and physical energy function

We perform a systematic study of the effects of sequence-independent backbone interactions and sequence-dependent side-chain interactions on protein folding using fragment assembly and physical energy function. Structures for ten proteins belonging to various structural classes are predicted only with Lennard-Jones interaction between backbone atoms. We find nativelike structures for beta proteins, suggesting that for proteins in this class, the global tertiary structures can be determined mainly by sequence-independent backbone interactions. On the other hand, for alpha proteins, nonlocal hydrophobic side-chain interaction is also required to obtain nativelike structures.     

Direct observation of the main-chain conformation of a combined liquid-crystal polymer

The conformation of the main-chain (backbone) of a combined main-chain/side-chain liquid crystalline polymer has been qualitatively determined by small angle neutron scattering in the oriented nematic, the smectic A and the smectic C phases. The polymer backbone presents only a weak anisotropy, of prolate shape, in the nematic and the smectic C phases. A stronger reorientation of the backbones in the direction of the applied magnetic field is measured for the S_A phase. However, this anisotropy remains small compared to the stretching of a main-chain liquid crystal polymer and the smectic structure results apparently from side-chain ordering. On the other hand, hydrodynamic measurements show that the combined polymer, in solvent, is as flexible as a polystyrene chain. This result is compatible with an explanation for the weak observed anisotropy.     

Measuring molecular order in poly(3-alkylthiophene) thin films with polarizing spectroscopies

We measured the molecular order of poly(3-alkylthiophene) chains in thin films before and after melting through the combination of several polarized photon spectroscopies: infrared (IR) absorption, variable angle spectroscopic ellipsometry (SE), and near-edge X-ray absorption fine structure (NEXAFS). The data from the various techniques can be uniformly treated in the context of the dielectric constant tensor epsilon for the film. The combined spectroscopies allow determination of the orientation distribution of the main-chain axis (SE and IR), the conjugated pi system normal (NEXAFS), and the side-chain axis (IR). We find significant improvement in the backbone order of the films after recrystallization of the material at temperatures just below the melting temperature. Less aggressive thermal treatments are less effective. IR studies show that the changes in backbone structure occur without significant alteration of the structure of the alkyl side chains. The data indicate that the side chains exhibit significant disorder for all films regardless of the thermal history of the sample.     

Phase and orientational ordering of low molecular weight rod molecules in a quenched liquid crystalline polymer matrix with mobile side chains

We study the phase diagram and orientational ordering of guest liquid crystalline (LC) rods immersed in a quenched host made of a liquid crystalline polymer (LCP) matrix with mobile side chains. The LCP matrix lies below the glass transition of the polymer backbone. The side chains are mobile and can align to the guest rod molecules in a plane normal to the local LCP chain contour. A field theoretic formulation for this system is proposed and the effects of the LCP matrix on LC ordering are determined numerically. We obtain simple analytical equations for the nematic/isotropic phase diagram boundaries. Our calculation show a nematic-nematic (N/N) first order transition from a guest stabilized to a guest-host stabilized region and the possibility of a reentrant transition from a guest stabilized nematic region to a host only stabilized regime separated by an isotropic phase. A detailed study of thermodynamic variables and interactions on orientational ordering and phases is carried out and the relevance of our predictions to experiments and computer simulations is presented.     

The magnetic field and temperature dependences of proton spin-lattice relaxation in proteins

The nuclear magnetic relaxation dispersion profiles of lyophilized globular proteins were measured in the frequency range of 10 kHz-30 MHz at temperatures from 156 to 302 K. The existent theory of proton relaxation in immobilized protein systems was critically tested and expended to include contributions of rapid motions of protein side-chain groups. The new theory takes into account the strong coupling between the side-chain protons and the protein backbone, when correlation function cannot be written as a product of the contributions. The measurements showed that while the relaxation rate constant of the protein backbone protons is a linear function of the absolute temperature the side-chain groups exhibit an exponential temperature dependence corresponding to an activated process. Measurements carried out on simple homopolypeptides, polyglycine and polyalanine, provide strong support of the proposed new theory.     

An investigation of the smectic-isotropic transition in a side-chain liquid crystal polymer by synchrotron radiation x-ray diffraction

In this work we have used synchrotron x-radiation diffraction to follow in real-time the isotropic-smoetic phase transition of a side-chain liquid crystalline polymer. The analysis of the x-ray data indicated that the transition occurs within a very narrow biphasic region. As the transition takes place, the size of the smectic regions, as indicated by the width of the diffraction peaks, changed only very slightly before impingement, suggesting a two-dimensional growth pattern. A thermal investigation of the polymer paralleling the x-ray experiments was undertaken and a kinetic analysis applied to the resulting data. Qualitative agreement with a disk-like growth of the smectic regions was found. We have shown that the overall kinetics of the isotropic-smectic transition of a side-chain polymer can be studied by x-ray diffraction permitting the evaluation of several structural parameters. © 1993 John Wiley & Sons, Inc.     

Phase behavior of a substituted poly(paraphenylene): Poly(para-2,5-didecyl-p-phenylene)

The thermal behavior of poly(para-2,5-didecyl-p-phenylene) has been investigated by differential scanning calorimetry and real time X-ray diffraction. Poly(para-2,5-didecyl-p-phenylene) is a semicrystalline material that crystallizes in a layered structure. The system exhibits two thermal transitions in the investigated temperature range. The first one, occurring at lower temperatures, provokes a reduction of the layered spacing accompanied by an appreciable disordering of the lateral side chains. Above the first transition the material is shearable, highly viscous, and birefringent. Thus, we have associated this transition to the formation of a layered mesophase. The higher temperature transition exhibits a twofold endothermic DSC peak and is characterized by the disappearance of X-ray diffracted intensity. At temperatures above the second transition the system presents the characteristics of an isotropic melt. Consequently, we have associated this transition with the complete disordering of the polymeric backbones. By following an appropriate thermal treatment it has been shown that the twofold shape of the endotherm characterizing the higher temperature transition can be changed into a single endotherm. This effect has been interpreted as being due to the kinetics of main-chain ordering. This ordering seems to proceed by the initial growth of domains with a high level of order followed by the subsequent increase of these domains through the inclusion of less ordered material. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36 : 49–54, 1998     

Effect of side chains on competing pathways for beta-scission reactions of peptide-backbone alkoxyl radicals

High-level quantum chemistry calculations have been carried out to investigate beta-scission reactions of alkoxyl radicals located at the alpha-carbon of a peptide backbone. This type of alkoxyl radical may undergo three possible beta-scission reactions, namely C-C beta-scission of the backbone, C-N beta-scission of the backbone, and C-R beta-scission of the side chain. We find that the rates for the C-C beta-scission reactions are all very fast, with rate constants of the order 10(12) s(-1) that are essentially independent of the side chain. The C-N beta-scission reactions are all slow, with rate constants that range from 10(-0.7) to 10(-4.5) s(-1). The rates of the C-R beta-scission reactions depend on the side chain and range from moderately fast (10(7) s(-1)) to very fast (10(12) s(-1)). The rates of the C-R beta-scission reactions correlate well with the relative stabilities of the resultant side-chain product radicals (*R), as reflected in calculated radical stabilization energies (RSEs). The order of stabilities for the side-chain fragment radicals for the natural amino acids is found to be Ala < Glu < Gln approximately Leu approximately Met approximately Lys approximately Arg < Asp approximately Ile approximately Asn approximately Val < Ser approximately Thr approximately Cys < Phe approximately Tyr approximately His approximately Trp. We predict that for side-chain C-R beta-scission reactions to effectively compete with the backbone C-C beta-scission reactions, the side-chain fragment radicals would generally need an RSE greater than approximately 30 kJ mol(-1). Thus, the residues that may lead to competitive side-chain beta-scission reactions are Ser, Thr, Cys, Phe, Tyr, His, and Trp.     

Intrinsic energy landscapes of amino acid side-chains

Amino acid side-chain conformational properties influence the overall structural and dynamic properties of proteins and, therefore, their biological functions. In this study, quantum mechanical (QM) potential energy surfaces for the rotation of side-chain χ(1) and χ(2) torsions in dipeptides in the alphaR, beta, and alphaL backbone conformations were calculated. The QM energy surfaces provide a broad view of the intrinsic conformational properties of each amino acid side-chain. The extent to which intrinsic energetics dictates side-chain orientation was studied through comparisons of the QM energy surfaces with χ(1) and χ(2) free energy surfaces from probability distributions obtained from a survey of high resolution crystal structures. In general, the survey probability maxima are centered in minima of the QM surfaces as expected for sp(3) (or sp(2) for χ(2) of Asn, Phe, Trp, and Tyr) atom centers with strong variations between amino acids occurring in the energies of the minima indicating intrinsic differences in rotamer preferences. High correlations between the QM and survey data were found for hydrophobic side-chains except Met, suggesting minimal influence of the protein and solution environments on their conformational distributions. Conversely, low correlations for polar or charged side-chains indicate a dominant role of the environment in stabilizing conformations that are not intrinsically favored. Data also link the presence of off-rotamers in His and Trp to favorable interactions with the backbone. Results also suggest that the intrinsic energetics of the side-chains of Phe and Tyr may play important roles in protein folding and stability. Analyses on whether intrinsic side-chain energetics can influence backbone preference identified a strong correlation for residues in the alphaL backbone conformation. It is suggested that this correlation reflects the intrinsic instability of the alphaL backbone such that assumption of this backbone conformation is facilitated by intrinsically favorable side-chain conformations. Together our results offer a broad overview of the conformational properties of amino acid side-chains and the QM data may be used as target data for force field optimization.     

Magic-angle spinning solid-state NMR spectroscopy of the beta1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational analysis

Magic-angle spinning solid-state NMR (SSNMR) studies of the beta1 immunoglobulin binding domain of protein G (GB1) are presented. Chemical shift correlation spectra at 11.7 T (500 MHz 1H frequency) were employed to identify signals specific to each amino acid residue type and to establish backbone connectivities. High sensitivity and resolution facilitated the detection and assignment of every 15N and 13C site, including the N-terminal (M1) 15NH3, the C-terminal (E56) 13C', and side-chain resonances from residues exhibiting fast-limit conformational exchange near room temperature. The assigned spectra lend novel insight into the structure and dynamics of microcrystalline GB1. Secondary isotropic chemical shifts report on conformation, enabling a detailed comparison of the microcrystalline state with the conformation of single crystals and the protein in solution; the consistency of backbone conformation in these three preparations is the best among proteins studied so far. Signal intensities and line widths vary as a function of amino acid position and temperature. High-resolution spectra are observed near room temperature (280 K) and at <180 K, whereas resolution and sensitivity greatly degrade substantially near 210 K; the magnitude of this effect is greatest among the side chains of residues at the intermolecular interface of the microcrystal lattice, which we attribute to intermediate-rate translational diffusion of solvent molecules near the glass transition. These features of GB1 will enable its use as an excellent model protein not only for SSNMR methods development but also for fundamental studies of protein thermodynamics in the solid state.     

Simulated evolution of emergent chiral structures in polyalanine

The relationship between monomer chirality and polymer structure has been studied using both theoretical and experimental methods. Atomistic models, such as the ones employed in computational protein folding and design, can be used to study the relationship between monomer chirality and the properties of polypeptides. Using a simulated evolution approach that combines side-chain epimerization with backbone flexibility, we recapitulate the relationship between basic forces that drive secondary structure formation and sequence homochirality. Additionally, we find heterochiral motifs including a C-terminal helix capping interaction and stable helix-reversals that result in bent helix structures. Our studies show that simulated evolution of chirality with backbone flexibility can be a powerful tool in the design of novel heteropolymers with tuned stereochemical properties.     

Coupling and memory in liquid crystal elastomers

The polymer backbone of a side-chain liquid crystal polymer exhibits an anisotropic shape due to the coupling of the liquid crystal orientational order of the mesogenic side-chains to the backbone. The magnitude and sign of this coupling may be controlled by chemical design. The introduction of chemical cross-links in to such a system provides both a memory of the anisotropic organisation and a mechanism by which the microscopic anisotropy can be realised at a macroscopic level. We show how this anisotropic network structure yields new phenomena when electric or mechanical fields are applied.     

Isotropic to smectic-C phase transition in liquid-crystalline elastomers

A phenomenological model is developed to describe the isotropic-smectic-C phase transition in liquid-crystalline side-chain elastomers. We analyze the influence of external mechanical stress on the isotropic-smectic-C phase transition. While this phase transition is first order in low-molecular-weight materials, we show here that the order of this transition does not change in liquid-crystalline elastomers. The temperature dependence of the heat capacity and the nonlinear dielectric effect in the isotropic phase above the isotropic-smectic-C phase transition in liquid crystalline elastomers are calculated. The theoretical results are found to be in good agreement with experiment.     

Energy minimizations with a combination of two knowledge-based potentials for protein folding

New force fields that are both simple and accurate are needed for computationally efficient molecular simulation studies to give insight into the actual features of the protein folding process. In this work, we assess a force field based on a new combination of two coarse-grained potentials taken from the bibliography. These potentials have already been proved efficient in representing different types of interactions, namely the side-chain interactions and the backbone hydrogen bonds. Now we combine them weighing their contribution to the global energy with a very simplified parameterization. To assess this combination of potentials, we use our evolutionary method to carry out energy minimization experiments for a set of all-alpha, all-beta, and (alpha + beta) protein structures. Our results, based on the assembly of short rigid native fragments, suggest that this combination of potentials can be successfully employed in coarse-grained folding simulations.